Bead Incubation and Washing on a Droplet Actuator

ABSTRACT

The invention relates to bead incubating and washing on a droplet actuator. Methods for incubating magnetically responsive beads that are labeled with primary antibody, a sample (i.e., analyte), and secondary reporter antibodies on a magnet, on and off a magnet, and completely off a magnet are provided. Also provided are methods for washing magnetically responsive beads using shape-assisted merging of droplets. Also provided are methods for shape-mediated splitting, transporting, and dispensing of a sample droplet that contains magnetically responsive beads. The apparatuses and methods of the invention provide for rapid time to result and optimum detection of an analyte in an immunoassay.

2 RELATED APPLICATIONS

This application is a continuation of and incorporates by reference U.S.patent application Ser. No. 14/731,740, entitled “Bead Incubation andWashing on a Droplet Actuator” filed on Jun. 5, 2015, which is acontinuation of and incorporates by reference U.S. patent applicationSer. No. 14/466,193 (now U.S. Pat. No. 9,081,007 issued Jul. 14, 2015),entitled “Bead Incubation and Washing on a Droplet Actuator” filed onAug. 22, 2014, which is a continuation of and incorporates by referenceU.S. patent application Ser. No. 14/081,376 (now U.S. Pat. No. 8,846,410issued Sep. 30, 2014), entitled “Bead Incubation and Washing on aDroplet Actuator” filed on Nov. 15, 2013, which is a divisional of andincorporates by reference U.S. patent application Ser. No. 13/081,927(now U.S. Pat. No. 8,637,324 issued Jan. 28, 2014), entitled “BeadIncubation and Washing on a Droplet Actuator” filed on Apr. 7, 2011,which is a continuation-in-part of U.S. patent application Ser. No.11/639,531 (now U.S. Pat. No. 8,613,889 issued Dec. 24, 2013), entitled“Droplet-based washing” filed on Dec. 15, 2006. U.S. patent applicationSer. No. 14/466,193 (now U.S. Pat. No. 9,081,007 issued Jul. 14, 2015),is a continuation-in-part of U.S. patent application Ser. No. 11/639,736(now U.S. Pat. No. 7,439,014 issued Oct. 21, 2008), entitled“Droplet-Based Surface Modification and Washing” filed on Dec. 15, 2006;Ser. No. 12/113,385 (now U.S. Pat. No. 8,541,176 issued Sep. 24, 2013),entitled “Droplet-Based Surface Modification and Washing” filed on May1, 2008, the application of which is a divisional of and incorporates byreference U.S. patent application Ser. No. 11/639,736 (now U.S. Pat. No.7,439,014 issued Oct. 21, 2008); Ser. No. 12/615,609 (now U.S. Pat. No.8,313,895 issued Nov. 20, 2012), entitled “Droplet-Based SurfaceModification and Washing” filed on Oct. 10, 2009, the application ofwhich is a continuation of and incorporates by reference U.S. patentapplication Ser. No. 12/113,385 (now U.S. Pat. No. 8,541,176 issued Sep.24, 2013), which is a divisional of U.S. patent application Ser. No.11/639,736 (now U.S. Pat. No. 7,439,014 issued Oct. 21, 2008); and Ser.No. 12/615,666, entitled “Droplet-Based Surface Modification andWashing” filed on Nov. 10, 2009, the application of which is acontinuation of and incorporates by reference U.S. patent applicationSer. No. 12/113,385 (now U.S. Pat. No. 8,541,176 issued Sep. 24, 2013),which is a divisional of U.S. patent application Ser. No. 11/639,736(now U.S. Pat. No. 7,439,014 issued Oct. 21, 2008). U.S. patentapplication Ser. No. 11/639,531 (now U.S. Pat. No. 8,613,889 issued Dec.24, 2013) and Ser. No. 11/639,736 (now U.S. Pat. No. 7,439,014 issuedOct. 21, 2008) claim priority to and incorporate by reference relatedprovisional U.S. Provisional Patent Application No. 60/745,058, entitled“Filler Fluids for Droplet-Based Microfluidics” filed on Apr. 18, 2006;60/745,039, entitled “Apparatus and Methods for Droplet-Based BloodChemistry,” filed on Apr. 18, 2006; 60/745,043, entitled “Apparatus andMethods for Droplet-Based PCR,” filed on Apr. 18, 2006; 60/745,059,entitled “Apparatus and Methods for Droplet-Based Immunoassay,” filed onApr. 18, 2006; 60/745,914, entitled “Apparatus and Method forManipulating Droplets with a Predetermined Number of Cells” filed onApr. 28, 2006; 60/745,950, entitled “Apparatus and Methods of SamplePreparation for a Droplet Microactuator,” filed on Apr. 28, 2006;60/746,797 entitled “Portable Analyzer Using Droplet-BasedMicrofluidics,” filed on May 9, 2006; 60/746,801, entitled “Apparatusand Methods for Droplet-Based Immuno-PCR,” filed on May 9, 2006;60/806,412, entitled “Systems and Methods for Droplet MicroactuatorOperations,” filed on Jun. 30, 2006; and 60/807,104, entitled “Methodand Apparatus for Droplet-Based Nucleic Acid Amplification,” filed onJul. 12, 2006.

Additionally, U.S. patent application Ser. No. 13/081,927 (now U.S. Pat.No. 8,637,324 issued Jan. 28, 2014), is a continuation of, claimspriority to, and incorporates by reference International PatentApplication Ser. No. PCT/US2009/059868, entitled “Bead Incubation AndWashing On A Droplet Actuator” International filing date of Oct. 7,2009, the application of which claims priority to U.S. ProvisionalPatent Application No. 61/103,302, filed on Oct. 7, 2008, entitled “BeadIncubation and Washing on a Droplet Actuator” and 61/122,791, filed onDec. 16, 2008, entitled “Bead Incubation and Washing on a DropletActuator,” the entire disclosures of which are incorporated herein byreference.

1 GRANT INFORMATION

This invention was made with government support under AI066590,HG003706, and CA114993 awarded by the National Institutes of Health. TheUnited States Government has certain rights in the invention.

3 FIELD OF THE INVENTION

The present invention provides methods and apparatuses for incubatingand washing magnetically responsive beads on a droplet actuator. Morespecifically the present invention provides methods for incubatingmagnetically responsive beads that are labeled with primary antibody, asample (i.e., analyte), and secondary reporter antibodies on a magnet,on and off a magnet, and completely off a magnet. The invention alsoprovides methods for washing magnetically responsive beads usingshape-assisted merging of droplets. The invention also provides methodsfor shape-mediated splitting, transporting, and dispensing of a sampledroplet that contains magnetically responsive beads. The methods of theinvention provide for rapid time to result and optimum detection of ananalyte in an immunoassay.

4 BACKGROUND OF THE INVENTION

Droplet actuators are used to conduct a wide variety of dropletoperations. A droplet actuator typically includes two substratesseparated by a gap. The substrates include electrodes for conductingdroplet operations. The gap between the substrates is typically filledwith a filler fluid that is immiscible with the fluid that is to besubjected to droplet operations. Droplet operations are controlled byelectrodes associated with one or both of the substrates. Dropletactuators are used in a variety of applications, including moleculardiagnostic assays, such as immunoassays where time to result is directlyaffected by the protocols used for each step of the assay. The most timeconsuming steps in an immunoassay are incubation and washing. “Time toresult” is directly affected by the protocols used for incubation, theduration of time for incubating the antibodies and the antigens, and theduration of time for incubating the substrate with sandwich beads, allof which may depend on the mixing efficiency within the droplets and thereaction and binding kinetics. The amount of washing required to obtainthe required sensitivity may also influence the total time to result forimmunoassays. There is a need for efficient incubation and washingprotocols for immunoassays on a droplet actuator that provide for rapidtime to result and optimum detection of an analyte.

5 BRIEF DESCRIPTION OF THE INVENTION

The present invention is directed to bead incubation and washing on adroplet actuator. The methods described herein may include providing adroplet including one or more magnetically responsive beads. The methodsmay include exposing the magnetically responsive beads in the droplet toa first region of a magnetic field capable of substantially attractingmagnetically responsive beads in the droplet. The methods may includeseparating the droplet from the first region of the magnetic field, themagnetically responsive beads remaining in the magnetic field. Themagnetically responsive beads may be separated from the droplet whileexposing the magnetically responsive beads in the droplet to a firstregion of a magnetic field and/or while the droplet is being separatedfrom the first region of the magnetic field. Exposing the magneticallyresponsive beads in the droplet to a first region of a magnetic fieldmay include transporting the droplet into the first region of themagnetic field and/or transporting the first region of the magneticfield into proximity with the magnetically responsive beads. Separatingthe droplet from the first region of the magnetic field may includetransporting the droplet away from the first region of the magneticfield and/or moving the first region of the magnetic field away from thedroplet.

In one embodiment, a method of incubating droplets having magneticallyresponsive beads is provided and comprises providing a droplet actuatorcomprising droplet operations electrodes arranged for conducting dropletoperations on a droplet operations surface and a magnet positionedrelative to the droplet operations surface such that a dropletcontrolled by one or more of the droplet operations electrodes may bepositioned within or away from a first region of the magnet's magneticfield capable of substantially attracting magnetically responsive beadsin the droplet. The method also comprises positioning a droplet havingmagnetically responsive beads therein at a location on the dropletoperations surface within the first region of the magnetic field to forma concentration of beads in the droplet; transporting the dropletthrough activation of selected droplet operations electrodes away fromthe first region of the magnetic field, thereby resuspending themagnetically responsive beads in the droplet; operating the dropletoperations electrodes to cause the droplet to split into two droplets,thereby redistributing the magnetically responsive beads; and operatingthe droplet operations electrodes to merge the two droplets into asingle droplet.

In another embodiment, a method of incubating droplets havingmagnetically responsive beads therein is provided and comprisesproviding a droplet actuator comprising droplet operations electrodesarranged for conducting droplet operations on a droplet operationssurface and a magnet positioned relative to the droplet operationssurface such that a droplet controlled by one or more of the dropletoperations electrodes may be positioned within or away from a firstregion of the magnet's magnetic field capable of substantiallyattracting magnetically responsive beads in the droplet. The method alsocomprises positioning a droplet having magnetically responsive beadstherein at a location within the first region of the magnetic field ofthe magnet to form a concentration of beads in the droplet; transportingthe droplet through activation of selected droplet operations electrodesaway from the first region of the magnetic field of the magnet toresuspend the magnetically responsive beads in the droplet; operatingthe droplet operations electrodes to cause the droplet to elongate andthen split into two droplets at a location away from the magnet; andoperating the droplet operations electrodes to merge the two dropletsinto a single droplet at a location away from the magnet, whereby thetransporting, splitting, and merging comprise an incubation cycle.

In yet another embodiment, a method of incubating droplets havingmagnetically responsive beads therein is provided and comprisesproviding a droplet actuator comprising droplet operations electrodesarranged for conducting droplet operations on a droplet operationssurface and a magnet positioned relative to the droplet operationssurface such that a droplet controlled by one or more of the dropletoperations electrodes may be positioned within or away from a firstregion of the magnet's magnetic field capable of substantiallyattracting magnetically responsive beads in the droplet. The methodfurther comprises positioning a droplet having magnetically responsivebeads therein on a droplet operations electrode, the droplet having afootprint approximately two times the area of a single dropletoperations electrode; transporting the droplet through activation ofselected droplet operations electrodes in one direction in a mannerelongating the droplet; and operating the droplet operations electrodesin a manner to cause the droplet to be transported in an oppositedirection to cause mixing and incubation within the droplet.

In a further embodiment, a method of washing magnetically responsivebeads for separating and removing unbound material is provided andcomprises providing a droplet actuator comprising droplet operationselectrodes arranged for conducting droplet operations on a dropletoperations surface and a magnet positioned relative to the dropletoperations surface such that a droplet controlled by one or more of thedroplet operations electrodes may be positioned within or away from afirst region of the magnet's magnetic field capable of substantiallyattracting magnetically responsive beads in the droplet. The methodfurther comprises positioning a droplet having magnetically responsivebeads therein to have a first region of the droplet within the firstregion of the magnetic field to form a concentration of beads; atanother end of the magnetic field, positioning a wash buffer dropletsuch that a first region of the wash buffer droplet is within the firstregion of the magnetic field; operating the droplet operationselectrodes to merge the droplet and the wash droplet to causeredistribution of beads; operating the droplet operation electrodes tocause the merged droplet to partially move away from the magnet, and tocause beads in the droplet to concentrate in the merged droplet; andoperating the droplet operations electrodes to split the merged dropletto form a supernatant droplet containing unbound reagents.

In a still further embodiment, a method of resuspending magneticallyresponsive beads between wash cycles is provided and comprises providinga droplet actuator comprising droplet operations electrodes arranged forconducting droplet operations on a droplet operations surface and amagnet positioned relative to the droplet operations surface such that adroplet controlled by one or more of the droplet operations electrodesmay be positioned within or away from a first region of the magnet'smagnetic field capable of substantially attracting magneticallyresponsive beads in the droplet. The method further comprisespositioning a droplet having magnetically responsive beads therein at alocation partially overlapping the first region of the magnetic field;transporting the droplet through activation of selected dropletoperations electrodes away from the first region of the magnetic field;operating the droplet operations electrodes to cause the droplet to movetowards the first region of the magnetic field; and repeating thetransporting and operating steps to cause sufficient resuspension ofbeads such that unbound material may be effectively removed insubsequent wash cycles.

In another embodiment, a droplet actuator device having a structure forconducting a bead washing protocol is provided and comprises an array ofdroplet operations electrodes configured to provide a plurality ofindividual wash lanes, and a single waste lane intersecting each one ofthe plurality of wash lanes; and waste wells associated at the end ofeach one of the plurality of wash lanes, and at the end of the singlewaste lane.

In yet another embodiment, a method of separating magneticallyresponsive beads from a droplet is provided and comprises providing adroplet actuator comprising droplet operations electrodes arranged forconducting droplet operations on a droplet operations surface and amagnet positioned relative to the droplet operations surface such that adroplet controlled by one or more of the droplet operations electrodesmay be positioned within or away from a first region of the magnet'smagnetic field capable of substantially attracting magneticallyresponsive beads in the droplet. The method further comprisespositioning a droplet having magnetically responsive beads thereinwithin the first region of the magnetic field of the magnet to cause themagnetically responsive beads to be attracted to the magnet, andactivating the droplet operations surface to cause the droplet to becircular in shape; operating the droplet operations surface to cause thedroplet to move away from the first region of the magnetic field to forma concentration of magnetically responsive beads in the droplet, and thedroplet operations surface being operated to cause the droplet to betransported away from the magnet one droplet operations electrode at atime, to cause the geometry of the droplet to be distorted; andcontinuing to transport the droplet away from the magnet to cause theconcentration of magnetically responsive beads to break away from thedroplet to result in a relatively small and highly concentratedmagnetically responsive bead droplet left behind and held immobilized bythe magnet.

In a further embodiment, a method of transporting magneticallyresponsive beads within droplets is provided and comprises providing adroplet actuator comprising droplet operations electrodes arranged forconducting droplet operations on a droplet operations surface and amagnet positioned relative to the droplet operations surface such that adroplet controlled by one or more of the droplet operations electrodesmay be positioned within or away from a first region of the magnet'smagnetic field capable of substantially attracting magneticallyresponsive beads in the droplet. The method further comprisespositioning a droplet having magnetically responsive beads locatedtherein at a location wherein the droplet partially overlaps the magnet;operating the droplet operations electrodes to subject an edge of thedroplet nearest the magnet to both a magnetic force from the firstregion of the magnetic field and an electrowetting force from thedroplet operations electrodes, and to subject an edge of the dropletfurthest from the magnet only to an electrowetting force, to cause thedroplet to be transported away from the magnet while retaining themagnetically responsive beads within the droplet; and continuing totransport the droplet away from the magnet to cause the magneticallyresponsive beads to be redistributed within the droplet.

In a still further embodiment, a method of separating beads from adroplet onto a magnet is provided and comprises providing a dropletactuator comprising droplet operations electrodes arranged forconducting droplet operations on a droplet operations surface and amagnet positioned relative to the droplet operations surface such that adroplet controlled by one or more of the droplet operations electrodesmay be positioned within or away from a first region of the magnet'smagnetic field capable of substantially attracting magneticallyresponsive beads in the droplet. The method further comprisespositioning a droplet having magnetically responsive beads thereinwithin the first region of the magnetic field of the magnet to cause thebeads to be attracted to the magnet, and activating the dropletoperations surface in a manner to cause the droplet to take an elongateshape; operating the droplet operations surface to activate oneelectrode at a time, to cause the droplet to move away from the magnet,and thereby cause the geometry of the droplet to be distorted; andcontinuing to operate the droplet operations surface to transport thedroplet further away from the magnet and inactivating an electrodeintermediate to the droplet to cause the droplet to split into asupernatant droplet and a smaller droplet that has the magneticallyresponsive beads therein.

In another embodiment, a droplet actuator structure for extracting DNAfrom a sample is provided and comprises at least six on-actuatorreservoirs interconnected for storing and dispensing different reagentsonto the droplet actuator; and the reservoirs interconnected throughpaths of droplet operations electrodes, including at least two pathshaving magnets associated therewith, and a bead collection reservoirconnected to the six on-actuator reservoirs through the dropletoperations electrodes paths.

In yet another embodiment, a method of extracting DNA from whole bloodis provided and comprises using a droplet actuator comprising at leastsix on-actuator reservoirs interconnected for storing and dispensingdifferent reagents onto the droplet actuator; and the reservoirsinterconnected through paths of droplet operations electrodes, includingat least two paths having magnets associated therewith, and a beadcollection reservoir connected to the six on-actuator reservoirs throughthe droplet operations electrodes paths. The method further comprisesdispensing a droplet of magnetically responsive beads suspended in alysis buffer from a first of the six on-actuator reservoirs, andtransporting the droplet through the droplet operations electrodes to aspecific location having one of the magnets associated with thelocation, to hold the magnetically responsive beads within the dropletthereon; dispensing droplets of whole blood from a second reservoir andlysis buffer from a third reservoir into a fourth mixing reservoir to bemixed therein to form a cell lysate; dispensing droplets of the celllysate across the magnetically responsive beads in succession andremoving supernatant from the droplets while holding the magneticallyresponsive beads; dispensing wash droplets from at least a fifthreservoir to wash the magnetically responsive beads to remove celldebris; and eluting and collecting DNA captured on the magneticallyresponsive beads at the bead collection reservoir.

In a further embodiment, a method of detecting a component in a sampleis provided and comprises providing a droplet actuator comprisingdroplet operations electrodes arranged for conducting droplet operationson a droplet operations surface; a magnet positioned related to thedroplet operations surface such that a droplet controlled by one of moredroplet operations electrodes may be positioned within or away from afirst region of the magnet's magnetic field capable of substantiallyattracting magnetically responsive beads in the droplet; and a washreservoir at one end of the arrangement of droplet operationselectrodes. The method further comprises positioning a droplet havingmagnetically responsive beads located therein, the magneticallyresponsive beads being coated with an antibody having an affinity for aspecific target antigen, away from the magnet; operating the dropletoperations surface in a manner to repeatedly transport the droplet backand forth, away from the magnet, in a manner to provide sufficientresuspension and mixing of the magnetically responsive beads forantibody and antigen binding; operating the droplet operations surfacein a manner to transport the droplet to a location within the firstregion of the magnetic field, and splitting off a supernatant dropletfrom the droplet by selectively operating the droplet operationssurface, and retaining the magnetically responsive beads at the magnet;operating the droplet operations electrodes to transport a reagentdroplet to the droplet operations electrode in the first region of themagnetic field to merge the reagent droplet with the droplet containingthe magnetically responsive beads, and transporting the merged dropletback and forth, at a location away from the magnet, to cause incubation;and transporting the merged droplet through operation of the dropletoperations electrodes to the droplet operations electrode at the magnetand splitting off a supernatant droplet through operation of the dropletoperations electrodes.

6 DEFINITIONS

As used herein, the following terms have the meanings indicated.

“Activate” with reference to one or more electrodes means effecting achange in the electrical state of the one or more electrodes whichresults in a droplet operation.

“Bead,” with respect to beads on a droplet actuator, means any bead orparticle that is capable of interacting with a droplet on or inproximity with a droplet actuator. Beads may be any of a wide variety ofshapes, such as spherical, generally spherical, egg shaped, disc shaped,cubical and other three dimensional shapes. The bead may, for example,be capable of being transported in a droplet on a droplet actuator orotherwise configured with respect to a droplet actuator in a mannerwhich permits a droplet on the droplet actuator to be brought intocontact with the bead, on the droplet actuator and/or off the dropletactuator. Beads may be manufactured using a wide variety of materials,including for example, resins, and polymers. The beads may be anysuitable size, including for example, microbeads, microparticles,nanobeads and nanoparticles. In some cases, beads are magneticallyresponsive; in other cases beads are not significantly magneticallyresponsive. For magnetically responsive beads, the magneticallyresponsive material may constitute substantially all of a bead or onecomponent only of a bead. The remainder of the bead may include, amongother things, polymeric material, coatings, and moieties which permitattachment of an assay reagent. Examples of suitable magneticallyresponsive beads are described in U.S. Patent Publication No.2005-0260686, entitled, “Multiplex flow assays preferably with magneticparticles as solid phase,” published on Nov. 24, 2005, the entiredisclosure of which is incorporated herein by reference for its teachingconcerning magnetically responsive materials and beads. The fluids mayinclude one or more magnetically responsive and/or non-magneticallyresponsive beads. Examples of droplet actuator techniques forimmobilizing magnetically responsive beads and/or non-magneticallyresponsive beads and/or conducting droplet operations protocols usingbeads are described in U.S. patent application Ser. No. 11/639,566,entitled “Droplet-Based Particle Sorting,” filed on Dec. 15, 2006; U.S.Patent Application No. 61/039,183, entitled “Multiplexing Bead Detectionin a Single Droplet,” filed on Mar. 25, 2008; U.S. Patent ApplicationNo. 61/047,789, entitled “Droplet Actuator Devices and DropletOperations Using Beads,” filed on Apr. 25, 2008; U.S. Patent ApplicationNo. 61/086,183, entitled “Droplet Actuator Devices and Methods forManipulating Beads,” filed on Aug. 5, 2008; International PatentApplication No. PCT/US2008/053545, entitled “Droplet Actuator Devicesand Methods Employing Magnetic Beads,” filed on Feb. 11, 2008;International Patent Application No. PCT/US2008/058018, entitled“Bead-based Multiplexed Analytical Methods and Instrumentation,” filedon Mar. 24, 2008; International Patent Application No.PCT/US2008/058047, “Bead Sorting on a Droplet Actuator,” filed on Mar.23, 2008; and International Patent Application No. PCT/US2006/047486,entitled “Droplet-based Biochemistry,” filed on Dec. 11, 2006; theentire disclosures of which are incorporated herein by reference.

“Droplet” means a volume of liquid on a droplet actuator that is atleast partially bounded by filler fluid. For example, a droplet may becompletely surrounded by filler fluid or may be bounded by filler fluidand one or more surfaces of the droplet actuator. Droplets may, forexample, be aqueous or non-aqueous or may be mixtures or emulsionsincluding aqueous and non-aqueous components. Droplets may take a widevariety of shapes; nonlimiting examples include generally disc shaped,slug shaped, truncated sphere, ellipsoid, spherical, partiallycompressed sphere, hemispherical, ovoid, cylindrical, and various shapesformed during droplet operations, such as merging or splitting or formedas a result of contact of such shapes with one or more surfaces of adroplet actuator.

“Droplet Actuator” means a device for manipulating droplets. Forexamples of droplets, see U.S. Pat. No. 6,911,132, entitled “Apparatusfor Manipulating Droplets by Electrowetting-Based Techniques,” issued onJun. 28, 2005 to Pamula et al.; U.S. patent application Ser. No.11/343,284, entitled “Apparatuses and Methods for Manipulating Dropletson a Printed Circuit Board,” filed on filed on Jan. 30, 2006; U.S. Pat.No. 6,773,566, entitled “Electrostatic Actuators for Microfluidics andMethods for Using Same,” issued on Aug. 10, 2004 and U.S. Pat. No.6,565,727, entitled “Actuators for Microfluidics Without Moving Parts,”issued on Jan. 24, 2000, both to Shenderov et al.; Pollack et al.,International Patent Application No. PCT/US2006/047486, entitled“Droplet-Based Biochemistry,” filed on Dec. 11, 2006, the disclosures ofwhich are incorporated herein by reference. Methods of the invention maybe executed using droplet actuator systems, e.g., as described inInternational Patent Application No. PCT/US2007/009379, entitled“Droplet manipulation systems,” filed on May 9, 2007. In variousembodiments, the manipulation of droplets by a droplet actuator may beelectrode mediated, e.g., electrowetting mediated or dielectrophoresismediated.

“Droplet operation” means any manipulation of a droplet on a dropletactuator. A droplet operation may, for example, include: loading adroplet into the droplet actuator; dispensing one or more droplets froma source droplet; splitting, separating or dividing a droplet into twoor more droplets; transporting a droplet from one location to another inany direction; merging or combining two or more droplets into a singledroplet; diluting a droplet; mixing a droplet; agitating a droplet;deforming a droplet; retaining a droplet in position; incubating adroplet; heating a droplet; vaporizing a droplet; condensing a dropletfrom a vapor; cooling a droplet; disposing of a droplet; transporting adroplet out of a droplet actuator; other droplet operations describedherein; and/or any combination of the foregoing. The terms “merge,”“merging,” “combine,” “combining” and the like are used to describe thecreation of one droplet from two or more droplets. It should beunderstood that when such a term is used in reference to two or moredroplets, any combination of droplet operations sufficient to result inthe combination of the two or more droplets into one droplet may beused. For example, “merging droplet A with droplet B,” can be achievedby transporting droplet A into contact with a stationary droplet B,transporting droplet B into contact with a stationary droplet A, ortransporting droplets A and B into contact with each other. The terms“splitting,” “separating” and “dividing” are not intended to imply anyparticular outcome with respect to size of the resulting droplets (i.e.,the size of the resulting droplets can be the same or different) ornumber of resulting droplets (the number of resulting droplets may be 2,3, 4, 5 or more). The term “mixing” refers to droplet operations whichresult in more homogenous distribution of one or more components withina droplet. Examples of “loading” droplet operations includemicrodialysis loading, pressure assisted loading, robotic loading,passive loading, and pipette loading. In various embodiments, thedroplet operations may be electrode mediated, e.g., electrowettingmediated or dielectrophoresis mediated.

“Filler fluid” means a fluid associated with a droplet operationssubstrate of a droplet actuator, which fluid is sufficiently immisciblewith a droplet phase to render the droplet phase subject toelectrode-mediated droplet operations. The filler fluid may, forexample, be a low-viscosity oil, such as silicone oil. Other examples offiller fluids are provided in International Patent Application No.PCT/US2006/047486, entitled, “Droplet-Based Biochemistry,” filed on Dec.11, 2006; and in International Patent Application No. PCT/US2008/072604,entitled “Use of additives for enhancing droplet actuation,” filed onAug. 8, 2008.

“Immobilize” with respect to magnetically responsive beads, means thatthe beads are substantially restrained in position in a droplet or infiller fluid on a droplet actuator. For example, in one embodiment,immobilized beads are sufficiently restrained in position to permitexecution of a splitting operation on a droplet, yielding one dropletwith substantially all of the beads and one droplet substantiallylacking in the beads.

“Magnetically responsive” means responsive to a magnetic field.“Magnetically responsive beads” include or are composed of magneticallyresponsive materials. Examples of magnetically responsive materialsinclude paramagnetic materials, ferromagnetic materials, ferrimagneticmaterials, and metamagnetic materials. Examples of suitable paramagneticmaterials include iron, nickel, and cobalt, as well as metal oxides,such as Fe₃O₄, BaFe₁₂O₁₉, CoO, NiO, Mn₂O₃, Cr₂O₃, and CoMnP.

“Washing” with respect to washing a magnetically responsive bead meansreducing the amount and/or concentration of one or more substances incontact with the magnetically responsive bead or exposed to themagnetically responsive bead from a droplet in contact with themagnetically responsive bead. The reduction in the amount and/orconcentration of the substance may be partial, substantially complete,or even complete. The substance may be any of a wide variety ofsubstances; examples include target substances for further analysis, andunwanted substances, such as components of a sample, contaminants,and/or excess reagent. In some embodiments, a washing operation beginswith a starting droplet in contact with a magnetically responsive bead,where the droplet includes an initial amount and initial concentrationof a substance. The washing operation may proceed using a variety ofdroplet operations. The washing operation may yield a droplet includingthe magnetically responsive bead, where the droplet has a total amountand/or concentration of the substance which is less than the initialamount and/or concentration of the substance. Other embodiments aredescribed elsewhere herein, and still others will be immediatelyapparent in view of the present disclosure.

The terms “top” and “bottom” are used throughout the description withreference to the top and bottom substrates of the droplet actuator forconvenience only, since the droplet actuator is functional regardless ofits position in space.

“Transporting into the magnetic field of a magnet,” “transportingtowards a magnet,” and the like, as used herein to refer to dropletsand/or magnetically responsive beads within droplets, is intended torefer to transporting into a region of a magnetic field capable ofsubstantially attracting magnetically responsive beads in the droplet.Similarly, “transporting away from a magnet or magnetic field,”“transporting out of the magnetic field of a magnet,” and the like, asused herein to refer to droplets and/or magnetically responsive beadswithin droplets, is intended to refer to transporting away from a regionof a magnetic field capable of substantially attracting magneticallyresponsive beads in the droplet, whether or not the droplet ormagnetically responsive beads is completely removed from the magneticfield. It will be appreciated that in any of such cases describedherein, the droplet may be transported towards or away from the desiredregion of the magnetic field, and/or the desired region of the magneticfield may be moved towards or away from the droplet. Reference to anelectrode, a droplet, or magnetically responsive beads being “within” or“in” a magnetic field, or the like, is intended to describe a situationin which the electrode is situated in a manner which permits theelectrode to transport a droplet into and/or away from a desired regionof a magnetic field, or the droplet or magnetically responsive beadsis/are situated in a desired region of the magnetic field, in each casewhere the magnetic field in the desired region is capable ofsubstantially attracting any magnetically responsive beads in thedroplet. Similarly, reference to an electrode, a droplet, ormagnetically responsive beads being “outside of” or “away from” amagnetic field, and the like, is intended to describe a situation inwhich the electrode is situated in a manner which permits the electrodeto transport a droplet away from a certain region of a magnetic field,or the droplet or magnetically responsive beads is/are situated awayfrom a certain region of the magnetic field, in each case where themagnetic field in such region is capable of substantially attracting anymagnetically responsive beads in the droplet.

When a liquid in any form (e.g., a droplet or a continuous body, whethermoving or stationary) is described as being “on”, “at”, or “over” anelectrode, array, matrix or surface, such liquid could be either indirect contact with the electrode/array/matrix/surface, or could be incontact with one or more layers or films that are interposed between theliquid and the electrode/array/matrix/surface.

When a droplet is described as being “on” or “loaded on” a dropletactuator, it should be understood that the droplet is arranged on thedroplet actuator in a manner which facilitates using the dropletactuator to conduct one or more droplet operations on the droplet, thedroplet is arranged on the droplet actuator in a manner whichfacilitates sensing of a property of or a signal from the droplet,and/or the droplet has been subjected to a droplet operation on thedroplet actuator.

7 BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, and 1E illustrate top views of an example of aregion of a droplet actuator and show a process of incubating beads on amagnet;

FIGS. 2A, 2B, 2C, 2D, and 2E illustrate top views of an example of aregion of a droplet actuator of FIGS. 1A through 1E and show a processof incubating droplets that include magnetically responsive beads on andoff a magnet;

FIGS. 3A, 3B, 3C, 3D, and 3E illustrate top views of an example of aregion of a droplet actuator and show a process of incubating dropletsthat include magnetically responsive beads completely off a magnet;

FIG. 4 shows a plot of a comparison of incubation time between on-magnetand off-magnet incubation protocols of FIG. 1 and FIG. 2, respectively,on immunoassay performance measured in chemiluminescence;

FIGS. 5A, 5B, 5C, 5D, and 5E show a top view of a region of a dropletactuator and a process of washing magnetically responsive beads usingshape-assisted merging of droplets;

FIG. 6 shows a plot of a comparison of washing protocols between slugshaped and circular shaped wash droplets on immunoassay performancemeasured in chemiluminescence;

FIGS. 7A, 7B, 7C, and 7D illustrate top views of an example of a regionof a droplet actuator of FIGS. 5A through 5E and show a process ofresuspending magnetically responsive beads between wash cycles;

FIGS. 8A and 8B show plots of a comparison of washing protocols of FIG.5 without resuspension cycles and with resuspension cycles,respectively;

FIG. 9 illustrates a top view of a region of a droplet actuator 900 thatincludes multiple waste wells;

FIGS. 10A, 10B, and 10C show a top view of a region of a dropletactuator and a process of separating beads from a sample droplet;

FIGS. 11A, 11B, and 11C show a top view of a region of a dropletactuator of FIGS. 10A through 10C and a process of transporting beadswithin a droplet;

FIGS. 12A, 12B, and 12C show a top view of a region of a dropletactuator of FIGS. 10A through 10C and another process of separatingbeads from a sample droplet onto a magnet;

FIGS. 13A and 13B show a comparison of bench top and droplet actuatorimmunoassay reagent ratios and a plot of reagent concentration versussignal strength, respectively, that provide for optimum droplet basedimmunoassay performance;

FIG. 14 shows a plot of the kinetics of a reaction between achemiluminescent substrate and ALP reporter on magnetically responsivebeads for Troponin I (TnI);

FIG. 15 is a top view of a droplet actuator that may be used forextracting DNA from a whole blood sample;

FIGS. 16A and 16B illustrate top views of an example of a portion of adroplet actuator and show a process of cytokine detection on a dropletactuator;

FIG. 17 shows a plot of two 5-point standard curves for cytokine IL-6;and

FIG. 18 shows a plot of two 6-point standard curves for cytokine TNF-α.

8 DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and apparatuses for incubatingand washing magnetically responsive beads on a droplet actuator. Morespecifically the present invention provides methods for incubatingmagnetically responsive beads that are labeled with primary antibody, asample (i.e., analyte), and secondary reporter antibodies on a magnet,on and off a magnet, and completely off a magnet. The invention alsoprovides methods for washing magnetically responsive beads usingshape-assisted merging of droplets. The invention also provides methodsfor shape-mediated splitting, transporting, and dispensing of a sampledroplet that contains magnetically responsive beads. The methods of theinvention provide for rapid time to result and optimum detection of ananalyte in an immunoassay.

In an alternative embodiment of the invention, a droplet actuator may beused to extract human genomic DNA from a sample.

8.1 Incubation Protocols

Incubation protocols on a droplet actuator are generally comprised oftransporting a droplet (e.g., a droplet that includes an antigen,primary capture antibodies conjugated to magnetically responsive beads,and secondary reporter antibodies) along a path of electrodes by use ofsplitting and merging operations that are inserted between transportcycles. Transporting, splitting, and merging the droplet ensures thatthe beads are well distributed (i.e., mixed) within the droplet. Anincubation cycle (e.g., transport, split, and merge) may be repeated twoor more times. The high mixing efficiency provided by a series ofincubation cycles provides for sufficient antigen-antibody binding.

Magnetically responsive beads have a tendency to settle and formaggregates due to gravity and/or continued exposure to strong magneticforces. These aggregates reduce the available surface area for bindingand slow down reaction kinetics and, consequently, the time to resultand sensitivity of the assay. Moreover, interstices in magneticallyresponsive bead aggregates can hold unbound species that leads toineffective washing. This may result in less sensitive assays andinaccuracies between assays due to differing amounts of unbound speciesheld in the interstices. Therefore, it is useful to keep the beadsdispersed or resuspended during incubation and in the steps immediatelyfollowing separation for further processing of the droplets away fromthe magnets. Resuspension of magnetically responsive beads withindroplets, akin to rigorous vortexing of bench scale systems, may beachieved by moving the bead droplet back and forth and exploiting theinherent circulatory flow patterns that are developed during droplettransport.

FIGS. 1A, 1B, 1C, 1D, and 1E illustrate top views of an example of aregion of a droplet actuator 100 and show a process of incubatingdroplets that include magnetically responsive beads on a magnet. Themethod of the invention of FIGS. 1A through 1E is an example of anincubation method wherein a droplet is transported into the magneticfield of a magnet and a series of split and merge droplet operations areperformed to resuspend the beads within the droplet. Because of themagnet field, resuspension of the beads is primarily in the X-Y todirection.

Droplet actuator 100 may include a path or array of droplet operationselectrodes 110 (e.g., electrowetting electrodes). A magnet 114 isarranged in close proximity to droplet operations electrodes 110. Inparticular, magnet 114 is arranged such that certain droplet operationselectrodes 100 (e.g., 3 droplet operations electrodes 110M) are withinthe magnetic field of magnet 114. Magnet 114 may, for example, be apermanent magnet or an electromagnet. Droplet actuator 100 may contain adroplet 118 that may be transported along droplet operations electrodes110 via electrowetting and upon which droplet operations may beperformed. Droplet 118 may, for example, be a 3× droplet, meaning thatits footprint is approximately 3 times the area of one dropletoperations electrode 110. Droplet 118 may, for example, include 1 partmagnetically responsive beads and 2 parts sample. Droplet 118 may, forexample, be a sample droplet that includes an analyte (e.g., an antigen)to be evaluated.

Droplet 118 may include one or more beads 122, which may be magneticallyresponsive beads. Beads 122 may have an affinity for certain targetsubstances, such as, for example, a certain type of cell, protein,nucleic acid and/or antigen. In one example, beads 122 are coated with aprimary antibody with affinity for a specific target antigen.

FIG. 1A shows a first step in a process of incubating droplets thatincludes magnetically responsive beads on a magnet. In this step,droplet 118 that has beads 122 therein is positioned adjacent to andoverlapping droplet operations electrodes 110M, which is within themagnetic field of magnet 114. Because beads 122 are magneticallyresponsive, a concentration of beads is formed at the side of droplet118 that is closest to magnet 114.

FIG. 1B shows another step in the process of incubating droplets thatinclude magnetically responsive beads on a magnet. In this step, droplet118 is transported via electrowetting to adjacent electrodes 110M andtakes on a slug-shaped geometry. Typically, two droplet operationselectrodes 110 may be used to transport a 3× droplet 118. Because beads122 are magnetically responsive, a concentration of beads 122 is formedat the bottom of droplet 118 (e.g., on the surface of droplet actuator100) and at the center of magnet 114. Elongation of droplet 118 to aslug geometry provides for sufficient flow of fluid within droplet 118to resuspend beads 122 in droplet 118.

FIG. 1C shows yet another step in the process of incubating dropletsthat include magnetically responsive beads on a magnet. In this step,droplet 118 is split near the central region of magnet 114 using dropletoperations to form, for example, two sample droplets. Splitting ofdroplet 118 provides for redistribution of beads 122 within the sampledroplets.

FIG. 1D shows yet another step in the process of incubating dropletsthat include magnetically responsive beads on a magnet. In this step,split droplet 118 is merged on magnet 114 using droplet operations toform a single droplet 118. The transporting, splitting, and mergingoperations of FIGS. 1B, 1C, and 1D comprise an incubation cycle. Severalincubation cycles may be performed to provide for resuspension andredistribution (i.e., mixing) of beads 122 in droplet 118.

FIG. 1E shows yet another step in the process of incubating dropletsthat include magnetically responsive beads on a magnet. In this step,magnet 114, which is, for example, an electromagnet, is not activated.Therefore, no magnetic field is generated by magnet 114 and beads 122 ofdroplet 118 have no attraction to magnet 114. Droplet 118 is transportedvia electrowetting to adjacent electrodes 110. Beads 122 are resuspendedin droplet 118.

FIGS. 2A, 2B, 2C, 2D, and 2E illustrate top views of an example of aregion of a droplet actuator 100 of FIGS. 1A through 1E and show aprocess of incubating, on and off a magnet, droplets that includemagnetically responsive beads. The method of the invention of FIGS. 2Athrough 2E is an example of an incubation method wherein a droplet istransported near the magnetic field of a magnet and then away from themagnet to perform a series of split and merge droplet operations thatare used to resuspend the beads within the droplet. Because the splitand merge operations are performed away from the magnet, resuspension ofthe beads is in the lateral X-Y, and vertical Z directions.

The steps shown in FIGS. 2A, 2B, 2C, 2D, and 2E are substantially thesame as those that are described in FIGS. 1A, 1B, 1C, 1D, and 1E exceptthat, instead of incubating droplet 118 on droplet operations electrodes110M, droplet incubation is performed adjacent to magnet 114 and awayfrom magnet 114 on droplet operations electrodes 110.

FIG. 2A shows a first step in a process of incubating droplets thatinclude magnetically responsive beads on and off a magnet. In this step,droplet 118 that has beads 122 therein is positioned adjacent to dropletoperations electrodes 110M, which is within the magnetic field of magnet114. Because beads 122 are magnetically responsive, a concentration ofbeads is formed at the side of droplet 118 that is closest to magnet114.

FIG. 2B shows another step in a process of incubating, on and off amagnet, droplets that include magnetically responsive beads. In thisstep, droplet 118 is transported via electrowetting away from themagnetic field of magnet 114. Beads 122 are sufficiently resuspended indroplet 118.

FIGS. 2C, 2D, and 2E show the process steps of droplet elongation (i.e.,formation of slug-shaped geometry), droplet splitting and dropletmerging, respectively, that are used to provide for sufficient flow offluid within droplet 118 to resuspend and redistribute beads 122 indroplet 118. Because the split and merge operations are performed awayfrom the magnet, resuspension of the beads is in the X, Y, and Zdirections.

FIGS. 3A, 3B, 3C, 3D, and 3E illustrate top views of an example of aregion of a droplet actuator 300 and show a process of incubatingdroplets that include magnetically responsive beads substantially out ofthe magnetic field of a magnet. The method of the invention of FIGS. 3Athrough 3E is an example of an incubation method wherein a series ofdroplet transport operations are used to resuspend the beads within thedroplet. Because the droplet operations are performed a sufficientdistance away from a magnet, resuspension of the beads is in the lateralX-Y, and vertical Z directions.

Droplet actuator 300 is substantially the same as droplet actuator 100of FIGS. 1A through 1E except that it is configured to support dropletoperations on a 2× droplet. In this embodiment, droplet 118 is a 2×droplet, meaning that its footprint is approximately 2 times the area ofone droplet operations electrode. Droplet 118 may, for example, include1 part magnetically responsive beads and 1 part sample. Alternatively,droplet 118 may include 1 part sample plus magnetically responsive beadsand 1 part secondary reporter antibody.

FIGS. 3A through 3E show the process steps of transporting droplet 118along a linear path of droplet operation electrodes 110 to providemixing of magnetically responsive beads 122. The use of a 2× dropletprovides several advantages over the use of a 3× droplet in a dropletactuator-based immunoassay. For example, mixing a 2× droplet using twodroplet operations electrodes is more efficient than mixing a 3× dropletusing two droplet operations electrodes. The concentration ofmagnetically responsive beads is also higher in a 2× droplet than a 3×droplet. A higher concentration of magnetically responsive beadsprovides for increased binding rate. Because the incubation cycle of a2× droplet is substantially out of the magnetic field of magnet 114, thebinding efficiency is also increased. In addition, transport of a 3×droplet in proximity to a magnetic field via electrowetting using 2droplet operation electrodes may result in the formation of a tail inwhich magnetically responsive beads may aggregate. The formation of beadaggregates may result in reduced binding efficiency.

FIG. 4 shows a plot 400 of a comparison of incubation time betweenon-magnet and off-magnet incubation protocols on immunoassay performancemeasured in chemiluminescence. Data was generated using the incubationprotocols of FIG. 1 (on-magnet) and FIG. 2 (off-magnet). Immunoassayswere performed on a 300 nanoliter (nL) droplet that contained captureantibody magnetically responsive beads, alkaline phosphatase(ALP)-labeled reporter antibodies and 5 ng/mL Troponin I (TnI). Fourimmunoassays were performed on the magnet with incubation times of 72seconds (sec), 198 sec, 450 sec, and 630 sec. Four more immunoassayswere performed off the magnet with incubation times of 90 sec, 180 sec,252 sec, and 324 sec. Droplet operations were the same for eachincubation protocol.

As shown in FIG. 4, when incubation was performed on the magnet, thetime to reach saturation was almost doubled the time to reach saturationwhen compared to incubation performed off the magnet. The difference intime to reach saturation between the two protocols may occur because inthe presence of a magnet, the magnetically responsive beads areattracted to the surface of the droplet actuator and the recirculationpatterns produce within a droplet would resuspend the magneticallyresponsive beads only in the lateral plane (X-Y) direction. Whenincubation is performed off the magnet and a sufficient distance awayfrom the magnet (e.g., more than 2 droplet operations electrodes),resuspension of the magnetically responsive beads is in both the lateralplane (X-Y direction) and vertical plane (Z direction). Resuspension ofthe beads in the X, Y, and Z directions provides more surface area forbinding reactions and increased rate of reaction.

In another embodiment, an incubation protocol may include merging of acircular bead droplet on a magnet with two circular sample droplets.Mixing in the merged droplet is provided by moving the merged dropletback and forth on droplet operations electrodes while the magneticallyresponsive beads are immobilized on the magnet.

In yet another embodiment, an incubation protocol may include merging ofa circular bead droplet on a magnet with a 4×, 5× elongated(slug-shaped) sample droplet. Mixing in the merged slug-shaped dropletis provided by moving the merged droplet back and forth on dropletoperations electrodes while the magnetically responsive beads areimmobilized on the magnet.

8.2 Washing Protocols

Washing of magnetically responsive beads, where unbound molecules areseparated and removed, is one of the most critical steps in implementingan immunoassay in a digital microfluidic system. In some embodiments,washing is performed using a merge-and-split protocol, which is repeateduntil the unbound material is sufficiently depleted from the supernatantto permit accurate and precise detection.

FIGS. 5A, 5B, 5C, 5D, and 5E show a top view of a region of a dropletactuator 500 and a process of washing magnetically responsive beadsusing shape-assisted merging of droplets. The method of the invention ofFIGS. 5A through 5E is an example of a wash method wherein a wash bufferdroplet and a magnetically responsive bead droplet are elongated in aslug-shaped geometry and series of merge and split operations are usedto remove unbound material from a bead droplet. The merge and splitoperations provide for substantially complete fluid replacement ofunbound supernatant with wash buffer in the absence of mixing.

Droplet actuator 500 may include a path or array of droplet operationselectrodes 510 (e.g., electrowetting electrodes). A magnet 512 isarranged in close proximity to droplet operations electrodes 510. Inparticular, magnet 512 is arranged such that certain droplet operationselectrodes 510 (e.g., 3 droplet operations electrodes 510M) are withinthe magnetic field of magnet 512. Magnet 512 may, for example, be apermanent magnet or an electromagnet. Droplet actuator 500 may contain awash buffer droplet 516 and a bead droplet 514 that may be transportedalong droplet operations electrodes 510 via electrowetting and uponwhich droplet operations may be performed. Bead droplet 514 may, forexample, include a quantity of magnetically responsive beads 518 thatincludes bound antigen and reporter antibody (i.e.,antigen-antibody-reporter complex), and unbound material such as excessunbound reporter antibody.

Bead droplet 514 and wash buffer droplet 516 may, for example, be 2×droplets, meaning that their footprint is approximately 2 times the areaof one droplet operations electrode 510. Bead droplet 514 and washbuffer droplet 516 may be configured as slug-shaped droplets (i.e.,elongated droplets) by performing droplet operations on the 2× dropletsusing two active droplet operations electrodes 510. Because the excessdroplet volume is now spread over a second active droplet operationselectrode 510, the droplets are elongated and conform to the shape oftwo electrodes.

FIG. 5A shows a first step in a process of washing beads usingshape-assisted merging of droplets. In this step, bead droplet 514 thathas beads 518 therein is positioned such that one region of droplet 514is on a droplet operations electrodes MOM which is within the magneticfield of magnet 512 and a second region of droplet 514 is on an adjacentdroplet operations electrode 510. Because beads 518 are magneticallyresponsive, a concentration of beads is formed at the side of beaddroplet 514 that is closest to magnet 512. At the opposite end of magnet512, wash buffer droplet 516 is similarly positioned such that oneregion of wash buffer droplet 516 is on a droplet operations electrodesMOM which is within the magnetic field of magnet 512 and a second regionof droplet 516 is on an adjacent droplet operations electrode 510. Thetiming of the sequence of droplet operations for merging bead droplet514 and wash buffer 516 is such that bead droplet 514 is elongated to aslug-shaped geometry just as wash buffer droplet is positioned viaelectrowetting for merging with bead droplet 514.

FIG. 5B shows another step in a process of washing beads usingshape-assisted merging of droplets. In this step, bead droplet 514 andwashed droplet 516 are merged. Merging of bead droplet 514 and washdroplet 516 provides for redistribution of beads 518.

FIGS. 5C and 5D show another step in a process of washing beads usingshape-assisted merging of droplets. In this step, a slug of fluid 520 isextended away from magnet 512 by activating the contiguous dropletoperations electrodes 510 and inactivating the intermediate dropletoperations electrodes 510 outside the magnetic field of magnet 512.Fluid 520 includes wash buffer from wash buffer droplet 516 and unboundreagents from bead droplet 514. As fluid 520 is extended, beads 518remain concentrated on magnet 512.

FIG. 5E shows yet another step in a process of washing beads usingshape-assisted merging of droplets. In this step, a fluid 520 is splitusing droplet operations to form supernatant droplet 522. Supernatantdroplet 522 includes unbound reagents such as unbound reporter antibodyfrom bead droplet 514. Supernatant droplet 522 is typically discarded ina waste well (not shown). FIGS. 5A through 5E show the set of dropletoperations that comprise a wash cycle. Several wash cycles may beperformed to provide for sufficient removal of unbound material.

In an alternative embodiment, a washing protocol may use a wash dropletand a bead droplet that are circular in shape. A circular shape of adroplet may, for example, be obtained by performing droplet operationson a 2× wash droplet and a 2× bead droplet using only one dropletoperations electrode each. Because a 2× droplet (i.e., footprint isapproximately 2 times the area of one droplet operations electrode) ismuch larger than a single droplet operations electrode, the droplettakes a more rounded shape.

FIG. 6 shows a plot 600 of a comparison of washing protocols betweenslug shaped and circular shaped wash droplets on immunoassay performancemeasured in chemiluminescence. In both protocols, incubation wasperformed using the off-magnet incubation protocol of FIG. 2 for 3minutes. Each wash cycle takes about 10 seconds in a slug-based protocolof FIG. 5 and about 14 seconds in a circular droplet protocol.

As shown is FIG. 6, a washing protocol performed using slugs of fluid(or elongated droplets as shown in FIG. 5), wherein a 2× wash bufferdroplet and a 2× bead droplet were operated using two electrodes, asufficient wash level was achieved using fewer wash cycles when comparedto washing using circular shaped droplets. In a slug-based washingprotocol, mixing was minimized and the bulk of the unbound material fromthe supernatant was replaced with fresh wash buffer at each cycle. In acircular-droplet based protocol, mixing was ensured by operating the 2×droplets using only one electrode each. Also, the dispersion ofmagnetically responsive beads in the lateral plane (X-Y direction) washigher in the slug-based protocol when a fresh wash buffer droplet wasmerged with a bead droplet. Greater dispersion of magneticallyresponsive beads in the merged droplet enables any unbound antibodytrapped in the interstices to diffuse into the supernatant and be washedaway in subsequent washes. In this example, sufficient wash levels wereachieved in about 10 wash cycles using a slug-based washing protocolcompared to >18 wash cycles in a circular droplet based wash protocol.

The washing behavior has two distinct regimes, one regime where washingmay be very pronounced and the second where the washing may be subtle.In the slug based washing case, the washing is pronounced with each washcycle up to about 9 cycles and after that the effect of washing isalmost negligible. In the circular droplet protocol, the washing effectis pronounced until about the 15th wash; although the wash efficiency isless than that observed for the slug-based protocol. Washing is onlymarginally effective for the circular droplet protocol between about the15th and 18th washes with only a slight reduction in signal with eachcycle. This could happen because all the free unbound material may bewashed away in the first few cycles and after that washing only removesthe unbound material trapped between the beads.

FIGS. 7A, 7B, 7C, and 7D illustrate top views of an example of a regionof a droplet actuator 500 of FIGS. 5A through 5E and show a process ofresuspending magnetically responsive beads between wash cycles. Themethod of the invention of FIGS. 7A through 7D is an example of asequence in a wash method that uses a series of droplet resuspensioncycles to resuspend the magnetically responsive beads between washcycles such as a wash cycle shown in FIG. 5. The resuspension cyclesprovide sufficient resuspension of the beads such that unbound materialfrom the interstices of bead aggregates may be effectively removed.

FIG. 7A shows the first step in a process of resuspending magneticallyresponsive beads between wash cycles. In this step, bead droplet 514that includes magnetically responsive beads 518 is transported viaelectrowetting away from magnet 512 in the direction of arrow A.

FIGS. 7B, 7C, and 7D show the process steps of transporting bead droplet514 along a path of droplet operation electrodes 510 in the direction ofarrow A. Three transport operations are shown in FIGS. 7B, 7C, and 7D,but any number of transport operations may be used to comprise aresuspension cycle. Transporting of bead droplet 514 provides forsufficient resuspension of beads 518 such that unbound material from theinterstices of bead aggregates may be effectively removed in subsequentwash cycles.

A complete wash protocol may include a series of wash cycles, such asthe slug based wash cycles of FIG. 5, interspersed with a one or moreresuspension cycles of FIG. 7. Depending on the sensitivity of the assayrequired and the time to result requirement, any number of wash cyclesmay be interspersed with any number of resuspension cycles. For example,a complete wash protocol sequence may include, for example, four washcycles, four resuspension cycles, and four wash cycles. A complete washprotocol sequence ends at a wash cycle.

FIGS. 8A and 8B show plots of a comparison of washing protocols of FIG.5 without resuspension cycles and with resuspension cycles,respectively. As shown in FIG. 8A, a washing protocol in the absence ofone or more resuspension cycles provides an initial drop in signal aftera number of wash cycles (A). As the number of wash cycles increase (B),there is a further reduction in signal that may be due to loss ofunbound material from the interstices of bead aggregates.

As shown in FIG. 8B, a washing protocol that includes one or moreresuspension cycles provides more efficient removal of unbound materialto a near zero level using fewer numbers of wash cycles (A).

FIG. 9 illustrates a top view of a region of a droplet actuator 900 thatincludes multiple waste wells. In this embodiment, multiple waste wellsare provided to improve the efficiency (e.g., time to result) of a beadwashing protocol such as a bead washing protocol shown in FIG. 5.

As illustrated, droplet actuator 900 includes an array of dropletoperations electrodes 910 (e.g., electrowetting electrodes) configuredto provide wash lanes 912 a, 912 b, 912 c, and 912 d, and a single wastelane 916. Wash lanes 912 a, 912 b, 912 c, and 912 d may include magnets914 a, 914 b, 914 c, and 914 d, respectively, and waste wells 920 a, 920b, 920 c, and 920 d, respectively. Waste lane 916 may include a wastewell 918.

In operation, droplet actuator 900 may be used to conduct a bead washingprotocol on four different samples in wash lanes 912 a through 912 d. Ina bead washing protocol, the supernatant droplet(s) that contain unboundmaterial, such as unbound antigen and secondary reporter antibody, istypically discarded in a waste well. In one example, a bead washingprotocol may use a single waste well 918. In this example, a singlewaste lane 916 that includes waste well 918 may be used to transportsupernatant (i.e., waste) droplets from wash lanes 912 a through 912 d.

In this example, supernatant (i.e., waste) droplets from wash lanes 912a through 912 d may be transported via electrowetting in the directionof Arrow A to wash lane 916. Individual supernatant droplets may then betransported in waste lane 916 in the direction of Arrow B to waste well918. Because waste lane 916 is common to wash lanes 912 a through 912 d,supernatant droplets must be transported serially (i.e., one afteranother).

In an alternative example, individual waste wells 920 a through 920 dmay be provided for each wash lane 912 a through 912 d, respectively. Inthis example, supernatant droplets may be transported simultaneously inthe direction of arrow C to individual waste wells 920 a through 920 d.Multiple, individual waste wells provide for increased efficiency (e.g.,time to result) in a washing protocol. Multiple waste wells also providefor a reduction in the number of droplet operations electrodes 910 thatare required to shuttle a supernatant droplet to a waste well. Areduction in the number of operations electrodes 910 that may be used totransport a supernatant droplet also provides for a reduction in thepotential for cross-contamination of subsequent droplets used in aprotocol.

8.3 Bead-Mediated Droplet Splitting

FIGS. 10A, 10B, and 10C show a top view of a region of a dropletactuator 1000 and a process of separating beads from a sample droplet.The method of the invention of FIGS. 10A through 10C is an example of amethod wherein magnetically responsive beads are split from a circularshaped droplet.

Droplet actuator 1000 may include a path or array of droplet operationselectrodes 1010 (e.g., electrowetting electrodes). A magnet 1014 isarranged in close proximity to droplet operations electrodes 1010. Inparticular, magnet 1014 is arranged such that certain droplet operationselectrodes 1010 (e.g., 3 droplet operations electrodes 1010M) are withinthe magnetic field of magnet 1014. Magnet 1014 may, for example, be apermanent magnet or an electromagnet.

Droplet actuator 1000 may contain a droplet 1016 that may be transportedalong droplet operations electrodes 1010 via electrowetting and uponwhich droplet operations may be performed. Droplet 1016 may include aquantity of beads 1020, which may be magnetically responsive beads. Anexample of a process of separating beads from a circular droplet mayinclude, but is not limited to, the following steps.

FIG. 10A shows a first step in a process of separating beads from acircular droplet. In this step, droplet 1016 that has beads 1020 thereinis positioned at a droplet operations electrode 1010M, which is withinthe magnetic field of magnet 1014. Because beads 1020 are magneticallyresponsive, beads 1020 are attracted to magnet 1014. Because a singledroplet operations electrode 1010M is active, droplet 1016 is circularin shape.

FIG. 10B shows another step in a process of separating beads from acircular droplet. In this step, droplet 1016 is transported viaelectrowetting away from droplet operations electrode 1010M and to theadjacent droplet operations electrode 1010. As droplet 1016 moves awayfrom droplet operations electrode 1010M, a concentration of beads 1020is formed at the side of droplet 1016 that is closest to magnet 1014.Because droplet 1016 is transported away from magnet 1014 one dropletoperations electrode 1010 at a time, the geometry of droplet 1016 may bedistorted (e.g., formation of a neck) by the concentration of beads 1020as droplet 1016 pulls away from magnet 1020.

FIG. 10C shows yet another step in the process of separating beads froma circular droplet. In this step, droplet 1016 is transported viaelectrowetting further away from droplet operations electrode 1010M andto a droplet operations electrode 1010 that is yet further away. Indoing so, the concentration of beads 1020 breaks away (snaps off) fromdroplet 1016. This occurs because one side (e.g., the side nearestmagnet 1014) of droplet 1016 is subjected to a magnetic force and theopposite side (e.g., the side farthest from magnet 1014) is subjected toelectrowetting force. When beads 1020 snap off of droplet 1016, arelatively small and highly concentrated bead droplet 1022 is leftbehind at droplet operations electrode 1010M and held immobilized bymagnet 1014.

A similar result can be achieved using a barrier that permits abead-containing droplet to be transported while restraining transport ofthe beads with the main body of the droplet.

FIGS. 11A, 11B, and 11C show a top view of a region of a dropletactuator 1000 of FIGS. 10A through 10C and a process of transportingbeads within a droplet. The method of the invention of FIGS. 11A through11C is an example of a method wherein magnetically responsive beads aretransported within an elongated droplet away from a magnetic force.

The steps shown in FIGS. 11A, 11B, and 11C are substantially the same asthose that are described in FIGS. 10A, 10B, and 10C except that, insteadof processing a 1× droplet via electrowetting using one active electrodeat a time, droplet 1016 is a slug-shaped 3× droplet that is processedusing three active electrodes for each droplet operation.

FIGS. 11A, 11B, and 11C show the process steps of transporting beads1020 within an elongated droplet 1016 away from magnet 1014. In thisexample, one side (e.g., the side nearest magnet 1014) of droplet 1016is subjected to both a magnetic force and electrowetting force and theopposite side (e.g., the side farthest from magnet 1014) is subjected toelectrowetting force. Because electrowetting force occurs on both sidesof droplet 1016, all of the fluid within the droplet is electrowettedand beads 1020 are retained within droplet 1016 during droplet transportaway from magnet 1014.

FIGS. 12A, 12B, and 12C show a top view of a region of a dropletactuator 1000 and a process of separating beads from a sample dropletonto a magnet. The method of the invention of FIGS. 12A through 12C isan example of a method wherein magnetically responsive beads are splitfrom an elongated droplet (e.g., 3× droplet) onto a magnet. In oneexample, the method of the invention of FIGS. 12A through 12C may beused to dispense magnetically responsive beads onto a magnet.

The steps shown in FIGS. 12A, 12B, and 12C are substantially the same asthose that are described in FIGS. 10A, 10B, and 10C except that, beads1020 are split from an elongated 3× droplet 1016. An example of aprocess of dispensing beads from an elongated droplet may include, butis not limited to, the following steps.

FIG. 12A shows a first step in a process of dispensing beads from anelongated droplet. In this step, droplet 1016 that has beads 1020therein is positioned at a droplet operations electrode 1010M, which iswithin the magnetic field of magnet 1014. Because beads 1020 aremagnetically responsive, beads 1020 are attracted to magnet 1014.Because three droplet operations electrodes 1010 are active, droplet1016 is elongated in shape.

FIG. 12B shows another step in a process of dispensing beads from anelongated droplet. In this step, droplet 1016 is transported away frommagnet 1014 via electrowetting using one active droplet operationselectrode 1010 at a time. As droplet 1016 moves away from dropletoperations electrode 1010M, a concentration of beads 1020 is formed atthe side of droplet 1016 that is closest to magnet 1014. Because droplet1016 is transported away from magnet 1014 one droplet operationselectrode 1010 at a time, the geometry of droplet 1016 may be distorted.

FIG. 12C shows yet another step in process of dispensing beads from anelongated droplet. In this step, droplet 1016 is transported viaelectrowetting further away from droplet operations electrode 1010M andto a droplet operations electrode 1010 that is yet further away. Oncethe sample droplet overlaps the droplet operation electrode 1010 onwhich droplet 1022 is to be formed, the intermediate electrode 1010 isdeactivated. In doing so, droplet 1016 is split into a supernatantdroplet 1022 and a smaller droplet 1016 that has beads 1020 therein.

8.4 Component Ratios

FIGS. 13A and 13B show a comparison of bench top and droplet actuatorimmunoassay reagent ratios and a plot of reagent concentration versussignal strength, respectively, that provide for optimum droplet basedimmunoassay performance.

As shown in FIG. 13A, the ratio of three components of an immunoassay,beads (i.e., capture antibody conjugated to beads), sample (e.g., serum,plasma), and secondary antibody (II^(∘)Ab) are provided. For a bench topimmunoassay, a typical ratio is 1 part beads (60 μL): ½ part sample (30μL): 1 part II^(∘)Ab (60 μL). A reagent ratio for a droplet actuatorbased immunoassay is typically ½ bead droplet (150 nL): 1 sample droplet(300 nL): 2 II^(∘)Ab droplets (600 nL). The use of fewer beads (i.e., ½bead droplet or ½ concentration of beads) in a droplet actuatorimmunoassay provides for increased efficiency of bead washing and asufficient reduction in non-specific binding of non-target analytes tothe capture beads. In addition, the concentration of secondary antibodyis the same in both bench top and droplet actuator immunoassays, but thevolume of secondary antibody solution is double in the droplet actuatorassay.

FIG. 13B shows the improvement in detection signal that is provided bythe use of 2 droplets of secondary antibody and 2 droplets of detectionsubstrate in a droplet actuator immunoassay.

8.5 Incubation of Magnetically Responsive Beads with ChemiluminescentSubstrate

Another parameter which may influence the time to result in animmunoassay is the generation of a signal during the incubation of achemiluminescent substrate with the washed magnetically responsive beadsthat contain the antigen-antibody complex.

FIG. 14 shows a plot 1400 of the kinetics of a reaction between achemiluminescent substrate and the ALP on magnetically responsive beadsfor Troponin I (TnI). Immunoassays were performed on TnI (100 ng/mL)using an on-magnet incubation protocol and a circular shaped dropletwashing protocol. As shown in FIG. 14, about 90% of the end point signalwas obtained in about 120 to about 130 seconds. For a lowerconcentration of the analyte, maximum signal was achieved in about <120seconds. Based on this data, for the type of substrate used, 2 minutesmay be selected as an optimum incubation time to generate maximum signalfor the chemiluminescence reaction. However, if the chemiluminescencereaction is observed to behave as a flash signal instead of a glowreaction, the 2 minute incubation may be reduced to about a few seconds.

8.6 Rapid Immunoassays

Using optimized protocols for incubation and washing, a full immunoassaywas performed on TnI (5 ng/mL). Magnetically responsive beads wereincubated with capture antibody, analyte and secondary antibody labeledwith ALP reporter using an off-magnet incubation protocol as shown inFIG. 3. Subsequently, ten slug-based washes were performed to remove theunbound material from the supernatant (wash time approximately 2minutes). The droplet with washed magnetically responsive beads with theantigen-antibody complex was mixed with one droplet of achemiluminescent substrate and incubated for 2 minutes. The end pointchemiluminescence was detected using a photon counter. In this example,the total time to result was approximately 10 minutes per immunoassay.

8.7 Protocol for Droplet Actuator Extraction of Human Genomic DNA

In an alternative embodiment of the invention, a droplet actuator may beused to extract human genomic DNA from a sample.

FIG. 15 is a top view of a droplet actuator 1500 that may be used forextracting DNA from a whole blood sample. The droplet actuator 1500includes six on-actuator reservoirs, each with a capacity of 2 μL, whichmay be used for storing and dispensing different reagents. A typicalprotocol for DNA extraction on a droplet actuator may include thefollowing steps.

In a first step, a droplet of magnetically responsive beads, such asparamagnetic to Dynabeads® DNA Direct Universal from Dynal Biotech (1.05μm diameter), suspended in a lysis buffer are dispensed from an on-chipreservoir and transported via electrowetting to a specific location onthe chip. The beads, which are magnetically responsive, are held by apermanent magnet placed underneath the chip.

In another step, droplets of whole blood are dispensed from a reservoirand mixed with droplets of lysis buffer (containing 10 M NaOH) dispensedfrom another on-chip reservoir, into a mixing reservoir in the ratio of1:6 and mixed for about 10 seconds. Mixing was performed by dispensing adroplet and then merging the droplet back into the reservoir.

In another step, droplets of the cell lysate were then transportedacross the DNA capture beads in succession and the supernatant waspinched off while holding the beads.

In another step, droplets of wash buffer stored in separate on-chipreservoirs were then used to wash the beads to remove cell debris.

In another step, purified genomic DNA captured on the beads was theneluted and collected at the bead collection reservoir. The collected DNAcan then be amplified either on the chip as part of an integratedsample-to-answer chip or in a commercial thermocycler for further DNAprocessing or diagnostic applications.

8.8 Operation Fluids

For examples of fluids that may be subjected to droplet operations usingthe approach of the invention, see the patents listed in section 6,especially

International Patent Application No. PCT/US2006/047486, entitled,“Droplet-Based Biochemistry,” filed on Dec. 11, 2006. In someembodiments, the fluid includes a biological sample, such as wholeblood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum,cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion,serous fluid, synovial fluid, pericardial fluid, peritoneal fluid,pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastricfluid, intestinal fluid, fecal samples, fluidized tissues, fluidizedorganisms, biological swabs, biological washes, liquids with cells,tissues, multicellular organisms, single cellular organisms, protozoa,bacteria, fungal cells, viral particles, organelles. In someembodiments, the fluid includes a reagent, such as water, deionizedwater, saline solutions, acidic solutions, basic solutions, detergentsolutions and/or buffers. In some embodiments, the fluid includes areagent, such as a reagent for a biochemical protocol, such as a nucleicacid amplification protocol, an affinity-based assay protocol, asequencing protocol, and/or a protocol for analyses of biologicalfluids.

The fluids may include one or more magnetically responsive and/ornon-magnetically responsive beads. Examples of droplet actuatortechniques for immobilizing magnetically responsive beads and/ornon-magnetically responsive beads are described in the foregoinginternational patent applications and in Sista, et al., U.S. PatentApplication No. 60/900,653, entitled “Immobilization ofMagnetically-responsive Beads During Droplet Operations,” filed on Feb.9, 2007; Sista et al., U.S. Patent Application No. 60/969,736, entitled“Droplet Actuator Assay Improvements,” filed on Sep. 4, 2007; and Allenet al., U.S. Patent Application No. 60/957,717, entitled “Bead WashingUsing Physical Barriers,” filed on Aug. 24, 2007, the entire disclosuresof which is incorporated herein by reference.

8.9 Example: Cytokine Immunoassay on a Droplet Actuator

FIGS. 16A and 16B illustrate top views of an example of a portion of adroplet actuator 1600 and show a process of cytokine detection on adroplet actuator. All steps involved in the immunoassay, includingsample and reagent aliquoting, incubation with antibodies, bead washing,and enzymatic detection, are fully automated and under software control.

Droplet actuator 1600 may include a path or array of droplet operationselectrodes 1610 (e.g., electrowetting electrodes) and a wash reservoir1612. A magnet 1614 is arranged in close proximity to droplet operationselectrodes 1610. In particular, magnet 1614 is arranged such that acertain droplet operations electrode 1610 (e.g., droplet operationselectrode 1610M) is within the magnetic field thereof. Magnet 1614 maybe a permanent magnet or an electromagnet. Droplet actuator 1600 maycontain a droplet 1618 that may be transported along droplet operationselectrodes 1610 and upon which droplet operations may be performed.

Droplet 1618 may, for example, be a 3× droplet, meaning that itsfootprint is approximately 3 times the area of one droplet operationselectrode 1610. Droplet 1618 may, for example, include 1 partmagnetically responsive beads and 2 parts sample (e.g., an antigen to beevaluated).

Droplet 1618 may include one or more magnetically responsive beads 1622.Magnetically responsive beads 1622 are coated with a primary antibodythat has an affinity for a specific target antigen. In one example,magnetically responsive beads 1622 are coated with a primary antibodythat has an affinity for IL-6. In another example, magneticallyresponsive beads 1622 are coated with a primary antibody that has anaffinity for TNF-α.

An example of a process of cytokine detection on a droplet actuator mayinclude, but is not limited to, the following steps:

Step A of FIG. 16A shows a droplet 1618 that has magnetically responsivebeads 1622 therein and is positioned at a certain droplet operationselectrode 1610. In one example, droplet 1618 includes 1 part beads 1622and 2 parts sample.

Steps B and C of FIG. 16A show an incubation process, in which droplet1618 is repeatedly transported back and forth via droplet operations toadjacent electrodes 1610. Repeated transporting of droplet 1618 is usedduring incubation of beads 1622 and sample in order to providesufficient resuspension and mixing of magnetically responsive beads 1622for optimal antibody and antigen binding. Typically, two dropletoperations electrodes 1610 may be used to transport a 3× droplet 1618,which takes on a slug shaped geometry. Elongation of droplet 1618 to aslug shape provides for sufficient flow of fluid within droplet 1618 toresuspend magnetically responsive beads 1622 therein. In one example,droplet 1618 may be incubated for 6 minutes using 2 droplet operationselectrodes 1610 and transporting droplet 1618 over a span of 8electrodes at a switching speed of 5 Hertz (Hz).

Step D of FIG. 16A shows droplet 1618 that has magnetically responsivebeads 1622 therein transported to droplet operations electrode 1610M. Asupernatant droplet 1624 is split off using droplet operations. Becausemagnetically responsive beads 1622 are attracted to magnet 1614, theyare retained at magnet 1614. In one example, supernatant droplet 1624 isa 1× droplet and droplet 1618 is now a 2× droplet. Supernatant droplet1624 that includes unbound antigen (i.e., cytokine) is discarded (notshown).

Step E of FIG. 16A shows a reagent droplet 1628 that includes secondaryantibody transported via electrowetting to droplet operations electrode1610M. Reagent droplet 1628 is merged with droplet 1618 (i.e., a 2×droplet) using droplet operations to form, for example, a 3× droplet.

In one example, reagent droplet 1628 is a 1× droplet that includesbiotinylated secondary antibody that has an affinity to the targetantigen. The antigen target is captured by the primary antibody which isimmobilized on the beads. Merged droplet 1618 is incubated for 4 minutesusing droplet operations, as described in steps B and C. Following theincubation period, droplet 1618 is transported via electrowetting todroplet operations electrode 1610M and a 1× supernatant droplet is splitoff using droplet operations, as described in step D, in order to yielda 2× droplet 1618. The supernatant droplet (not shown) that includesunbound secondary antibody is discarded.

After incubation with the biotinylated secondary antibody, the beads mayin some embodiments be washed and then incubated with thestreptavidin-peroxidase. The entire complex thus consists ofbeads-primary antibody-antigen-secondaryantibody-streptavidin-peroxidase. Streptavidin-peroxidase may besubstituted with streptavidin-alkaline phosphatase.

Step F of FIG. 16B shows a bead washing step, in which a wash droplet1630 is transported from wash reservoir 1612 along droplet operationselectrodes 1610 and across droplet 1618, which is retained at dropletoperations electrode 1610M. As wash droplet 1630 passes across magnet1614, droplet merge and split operations occur with droplet 1618 (i.e.,a 2× droplet). In one example, wash droplet 1630 is a 2× droplet thathas a slug geometry and the washing protocol is repeated 5 times.Following bead washing, a 1× supernatant droplet is split off fromdroplet 1618, as described in step D of FIG. 16A, in order to yield a 1×droplet 1618. The supernatant droplet (not shown) is discarded.

Step G of FIG. 16B shows one or more reagent droplets 1632 (e.g., 1632a, 1632 b) transported to droplet operations electrode 1610M. In oneexample, reagent droplet 1632 a that includes a blocking agent (e.g.,Synblock) and reagent droplet 1632 b that includes a streptavidin-enzymeconjugate (e.g., streptavidin-alkaline phosphatase (ALP) orstreptavidin-horseradish peroxidase) are transported to dropletoperations electrode 1610M and merged using droplet operations withdroplet 1618. Droplet 1618 is now a 3× droplet.

Merged droplet 1618 is incubated for 4 minutes using droplet operations,as described in steps B and C of FIG. 16A. Following the incubationperiod, droplet 1618 is transported to droplet operations electrode1610M and a supernatant droplet (i.e., a 1× droplet) is split off usingdroplet operations, as described in step D of FIG. 16A, in order toyield a 2× droplet 1618. The supernatant droplet (not shown) thatincludes unbound streptavidin-enzyme conjugate is discarded.

Droplet 1618 is subsequently washed, for example 15 times, as describedin step F of FIG. 16B. Following bead washing, a 1× supernatant dropletis split off droplet 1618, as described in step D of FIG. 16A, in orderto yield a 1× droplet 1618. The supernatant droplet (not shown) isdiscarded. Droplet 1618 that includes antibody-antigen sandwich is nowready for detection.

Step H of FIG. 16B shows droplet 1634 (1× droplet) that includes adetection substrate 1636 transported to droplet operations electrode1610M and merged using droplet operations with droplet 1618. Thedetection substrate 1636 is converted by the enzyme conjugate into afluorescent signal (product formation time about 1620 seconds). Thechemiluminescent signal is measured by a detector (not shown) in orderto determine the quantity of antigen that is present.

In some embodiments, wash buffer droplets may be transported across thedetection window following each chemiluminescent droplet to clean up thedetection window and the detection loop prior to the next detection.

8.9.1 IL-6 Results

In one embodiment, the method of the invention is used to detect IL-6.FIG. 17 shows a plot of two 5-point standard curves for cytokine IL-6.Data was generated using the cytokine detection protocol described inFIGS. 16A and 16B. In this example, two 5-point standard curves (0,0.05, 0.5, 5, and 25 ng/mL of IL-6) were obtained in 2 runs for IL-6performed on 2 separate droplet actuators.

8.9.2 TNF-α Results

In an alternative embodiment, the method of the invention is used todetect TNF-α. FIG. 18 shows a plot of two 6-point standard curves forcytokine TNF-α.

Data was generated using the cytokine detection protocol described inFIGS. 16A and 16B. In this example, two 6-point standard curves (0,0.01, 0.1, 1, 10, and 100 ng/mL of TNF-α) were obtained in 2 runs forTNF-α performed on 2 separate droplet actuators.

9 CONCLUDING REMARKS

The foregoing detailed description of embodiments refers to theaccompanying drawings, which illustrate specific embodiments of theinvention. Other embodiments having different structures and operationsdo not depart from the scope of the present invention. The term “theinvention” or the like is used with reference to certain specificexamples of the many alternative aspects or embodiments of theapplicants' invention set forth in this specification, and neither itsuse nor its absence is intended to limit the scope of the applicants'invention or the scope of the claims. This specification is divided intosections for the convenience of the reader only. Headings should not beconstrued as limiting of the scope of the invention. The definitions areintended as a part of the description of the invention. It will beunderstood that various details of the present invention may be changedwithout departing from the scope of the present invention. Furthermore,the foregoing description is for the purpose of illustration only, andnot for the purpose of limitation.

1-9. (canceled)
 10. A method of incubating droplets having magneticallyresponsive beads therein, the method comprising: (a) providing a dropletactuator comprising: (i) droplet operations electrodes arranged forconducting droplet operations on a droplet operations surface; and (ii)a magnet positioned relative to the droplet operations surface such thata droplet controlled by one or more of the droplet operations electrodesmay be positioned within or away from a first region of the magnet'smagnetic field capable of substantially attracting magneticallyresponsive beads in the droplet; (b) positioning a droplet havingmagnetically responsive beads therein at a location within the firstregion of the magnetic field of the magnet to form a concentration ofbeads in the droplet; (c) transporting the droplet through activation ofselected droplet operations electrodes away from the first region of themagnetic field of the magnet to resuspend the magnetically responsivebeads in the droplet; (d) operating the droplet operations electrodes tocause the droplet to elongate and then split into two droplets at alocation away from the magnet; and (e) operating the droplet operationselectrodes to merge the two droplets into a single droplet at a locationaway from the magnet, whereby the transporting, splitting, and mergingcomprise an incubation cycle.
 11. The method of claim 10, wherein instep (b) the concentration of magnetically responsive beads occurs at anedge of the droplet adjacent the magnet.
 12. The method of claim 10,wherein the droplet transported has a footprint approximately threetimes the area of a single droplet operations electrode.
 13. The methodof claim 10, wherein the droplet comprises a sample comprising a targetsubstance, and the magnetically responsive beads have an affinity forthe target substance.
 14. The method of claim 13, wherein the targetsubstances are at least one of cells, protein, nucleic acid, and anantigen.
 15. The method of claim 10, wherein the transporting,splitting, and merging operations are repeated a plurality of times. 16.A method of incubating droplets having magnetically responsive beadstherein, the method comprising: (a) providing a droplet actuatorcomprising: (i) droplet operations electrodes arranged for conductingdroplet operations on a droplet operations surface; and (ii) a magnetpositioned relative to the droplet operations surface such that adroplet controlled by one or more of the droplet operations electrodesmay be positioned within or away from a first region of the magnet'smagnetic field capable of substantially attracting magneticallyresponsive beads in the droplet; (b) positioning a droplet havingmagnetically responsive beads therein on a droplet operations electrode,the droplet having a footprint approximately two times the area of asingle droplet operations electrode; (c) transporting the dropletthrough activation of selected droplet operations electrodes in onedirection in a manner elongating the droplet; and (d) operating thedroplet operations electrodes in a manner to cause the droplet to betransported in an opposite direction to cause mixing and incubationwithin the droplet.
 17. The method of claim 16, wherein the dropletcomprises one part magnetically responsive beads and one part sample.18. The method of claim 16, wherein the droplet comprises one partmagnetically responsive beads and one part secondary reporterantibodies.
 19. The method of claim 16, conducted substantially outsideof the first region of the magnetic field.
 20. The method of claim 17,wherein the sample comprises a target substance, and the magneticallyresponsive beads have an affinity for the target substance.
 21. Themethod of claim 20, wherein the target substance is at least one ofcells, protein, nucleic acid, and an antigen.
 22. A method of washingmagnetically responsive beads for separating and removing unboundmaterial, the method comprising: (a) providing a droplet actuatorcomprising: (i) droplet operations electrodes arranged for conductingdroplet operations on a droplet operations surface; and (ii) a magnetpositioned relative to the droplet operations surface such that adroplet controlled by one or more of the droplet operations electrodesmay be positioned within or away from a first region of the magnet'smagnetic field capable of substantially attracting magneticallyresponsive beads in the droplet; (b) positioning a droplet havingmagnetically responsive beads therein to have a first region of thedroplet within the first region of the magnetic field to form aconcentration of beads; (c) at another end of the magnetic field,positioning a wash buffer droplet such that a first region of the washbuffer droplet is within the first region of the magnetic field; (d)operating the droplet operations electrodes to merge the droplet and thewash droplet to cause redistribution of beads; (e) operating the dropletoperation electrodes to cause the merged droplet to partially move awayfrom the magnet, and to cause beads in the droplet to concentrate in themerged droplet; and (f) operating the droplet operations electrodes tosplit the merged droplet to form a supernatant droplet containingunbound reagents.
 23. The method of claim 22, wherein in step (b) theconcentration of magnetically responsive beads in the droplet occurs atan edge of the droplet adjacent the first region of the magnetic field,and in step (e) the concentration of beads in the merged droplet occursat an edge of the merged droplet located at the magnet.
 24. The methodof claim 22, wherein in step (b) the droplet is positioned to have afirst region on a droplet operations electrode within the first regionof the magnetic field and a second region of the droplet on an adjacentdroplet operations electrode.
 25. The method of claim 22, wherein instep (c) the first region of the wash buffer droplet is positioned on adroplet operations electrode within the first region of the magneticfield, and the second region of the wash droplet is on an adjacentdroplet operations electrode.
 26. The method of claim 22, wherein thewash droplet and the sample droplet each have a footprint approximatelytwo times the area of one droplet operations electrode.
 27. The methodof claim 22, further comprising repeating the method a plurality oftimes to provide for sufficient removal of unbound material.
 28. Adroplet actuator device having a structure for conducting a bead washingprotocol, the structure comprising: (a) an array of droplet operationselectrodes configured to provide a plurality of individual wash lanes,and a single waste lane intersecting each one of the plurality of washlanes; and (b) waste wells associated at the end of each one of theplurality of wash lanes, and at the end of the single waste lane.
 29. Amethod of separating magnetically responsive beads from a droplet, themethod comprising: (a) providing a droplet actuator comprising: (i)droplet operations electrodes arranged for conducting droplet operationson a droplet operations surface; and (ii) a magnet positioned relativeto the droplet operations surface such that a droplet controlled by oneor more of the droplet operations electrodes may be positioned within oraway from a first region of the magnet's magnetic field capable ofsubstantially attracting magnetically responsive beads in the droplet;(b) positioning a droplet having magnetically responsive beads thereinwithin the first region of the magnetic field of the magnet to cause themagnetically responsive beads to be attracted to the magnet, andactivating the droplet operations surface to cause the droplet to becircular in shape; (c) operating the droplet operations surface to causethe droplet to move away from the first region of the magnetic field toform a concentration of magnetically responsive beads in the droplet,and the droplet operations surface being operated to cause the dropletto be transported away from the magnet one droplet operations electrodeat a time, to cause the geometry of the droplet to be distorted; and (d)continuing to transport the droplet away from the magnet to cause theconcentration of magnetically responsive beads to break away from thedroplet to result in a relatively small and highly concentratedmagnetically responsive bead droplet left behind and held immobilized bythe magnet.
 30. The method of claim 29, wherein in step (c) theconcentration of magnetically responsive beads occurs at an edge of thedroplet adjacent to the magnet.
 31. The method of claim 29, wherein instep (b) the droplet is positioned at a droplet operations electrodewithin the first region of the magnetic field of the magnet.
 32. Themethod of claim 29, wherein in step (c) the droplet is caused to movefrom the droplet operations electrode within the first region of themagnetic field partially to an adjacent droplet operations electrodeaway from the magnet.
 33. The method of claim 29, wherein the dropletcomprises a sample comprising a target substance, and the magneticallyresponsive beads comprise beads having an affinity for the targetsubstance.
 34. The method of claim 33, wherein the target substance isat least one of cells, protein, nucleic acid, and an antigen.
 35. Amethod of transporting magnetically responsive beads within droplets,the method comprising: (a) providing a droplet actuator comprising: (i)droplet operations electrodes arranged for conducting droplet operationson a droplet operations surface; and (ii) a magnet positioned relativeto the droplet operations surface such that a droplet controlled by oneor more of the droplet operations electrodes may be positioned within oraway from a first region of the magnet's magnetic field capable ofsubstantially attracting magnetically responsive beads in the droplet;(b) positioning a droplet having magnetically responsive beads locatedtherein at a location wherein the droplet partially overlaps the magnet;(c) operating the droplet operations electrodes to subject an edge ofthe droplet nearest the magnet to both a magnetic force from the firstregion of the magnetic field and an electrowetting force from thedroplet operations electrodes, and to subject an edge of the dropletfurthest from the magnet only to an electrowetting force, to cause thedroplet to be transported away from the magnet while retaining themagnetically responsive beads within the droplet; and (d) continuing totransport the droplet away from the magnet to cause the magneticallyresponsive beads to be redistributed within the droplet.
 36. The methodof claim 35, wherein the droplet comprises a sample comprising a targetsubstance, and the magnetically responsive beads comprise beads havingan affinity for the target substance.
 37. The method of claim 36,wherein the target substance is at least one of cells, protein, nucleicacid, and an antigen.
 38. A droplet actuator structure for extractingDNA from a sample, the structure comprising: (a) at least sixon-actuator reservoirs interconnected for storing and dispensingdifferent reagents onto the droplet actuator; and (b) the reservoirsinterconnected through paths of droplet operations electrodes, includingat least two paths having magnets associated therewith, and a beadcollection reservoir connected to the six on-actuator reservoirs throughthe droplet operations electrodes paths.
 39. A method of extracting DNAfrom whole blood by using the droplet actuator structure of claim 38,the method comprising: (a) dispensing a droplet of magneticallyresponsive beads suspended in a lysis buffer from a first of the sixon-actuator reservoirs, and transporting the droplet through the dropletoperations electrodes to a specific location having one of the magnetsassociated with the location, to hold the magnetically responsive beadswithin the droplet thereon; (b) dispensing droplets of whole blood froma second reservoir and lysis buffer from a third reservoir into a fourthmixing reservoir to be mixed therein to form a cell lysate; (c)dispensing droplets of the cell lysate across the magneticallyresponsive beads in succession and removing supernatant from thedroplets while holding the magnetically responsive beads; (d) dispensingwash droplets from at least a fifth reservoir to wash the magneticallyresponsive beads to remove cell debris; and (e) eluting and collectingDNA captured on the magnetically responsive beads at the bead collectionreservoir.
 40. The method of claim 39, wherein the magneticallyresponsive beads have an affinity for the DNA in the whole blood.
 41. Amethod of detecting a component in a sample, the method comprising: (a)providing a droplet actuator comprising: (i) droplet operationselectrodes arranged for conducting droplet operations on a dropletoperations surface; (ii) a magnet positioned related to the dropletoperations surface such that a droplet controlled by one of more dropletoperations electrodes may be positioned within or away from a firstregion of the magnet's magnetic field capable of substantiallyattracting magnetically responsive beads in the droplet; and (iii) awash reservoir at one end of the arrangement of droplet operationselectrodes; (b) positioning a droplet having magnetically responsivebeads located therein, the magnetically responsive beads being coatedwith an antibody having an affinity for a specific target antigen, awayfrom the magnet; (c) operating the droplet operations surface in amanner to repeatedly transport the droplet back and forth, away from themagnet, in a manner to provide sufficient resuspension and mixing of themagnetically responsive beads for antibody and antigen binding; (d)operating the droplet operations surface in a manner to transport thedroplet to a location within the first region of the magnetic field, andsplitting off a supernatant droplet from the droplet by selectivelyoperating the droplet operations surface, and retaining the magneticallyresponsive beads at the magnet; (e) operating the droplet operationselectrodes to transport a reagent droplet to the droplet operationselectrode in the first region of the magnetic field to merge the reagentdroplet with the droplet containing the magnetically responsive beads,and transporting the merged droplet back and forth, at a location awayfrom the magnet, to cause incubation; and (f) transporting the mergeddroplet through operation of the droplet operations electrodes to thedroplet operations electrode at the magnet and splitting off asupernatant droplet through operation of the droplet operationselectrodes.
 42. The method of claim 41, further comprising conductingthe method to detect cytokine.
 43. The method of claim 41, furthercomprising transporting a wash droplet from the wash reservoir throughoperation of the droplet operations electrodes, across the magnet tocause merging of the droplets, and splitting to yield a washed dropletwith beads therein, and discarding a resultant supernatant droplet. 44.The method of claim 41, further comprising transporting at least onereagent droplet to the droplet operations electrode in the first regionof the magnetic field, and merging the reagent droplet with the dropletthrough operation of the droplet operations electrodes, incubating themerged droplet away from the magnet, transporting the merged dropletfollowing incubation to the droplet operations electrode in the firstregion of the magnetic field, and splitting off a supernatant droplet.45. The method of claim 41, further comprising washing the droplet. 46.The method of claim 41, wherein the droplet is a sample droplet having atarget antigen as a sample therein.